Glyphosate (N-phosphonomethylglycine) is a widely used component in herbicides. Glyphosate inhibits 5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase, or EPSPS), which is involved in the synthesis of aromatic amino acids in plant cells. Inhibition of EPSPS effectively disrupts protein synthesis and thereby kills the affected plant cells. Because glyphosate is non-selective, it kills both weeds and crop plants. Thus it is useful with crop plants when one can modify the crop plants to be resistant to glyphosate, allowing the desirable plants to survive exposure to the glyphosate. Accordingly, there is a need to produce transgenic crop plants that are resistant to glyphosate.
Recombinant DNA technology has been used to isolate mutant EPSP synthases that are glyphosate-resistant. Such glyphosate-resistant mutant EPSP synthases can be transformed into plants and confer glyphosate-resistance upon the transformed plants. By way of example, a glyphosate tolerant gene was isolated from Agrobacterium strain CP4 as described in U.S. Pat. No. 5,633,435. This reference and all references cited are incorporated herein by reference.
Other glyphosate tolerant genes have been created through the introduction of mutations. These include those isolated by Comai and described at U.S. Pat. Nos. 5,094,945, 4,769,061 and 4,535,060. A single mutant has been utilized, as described in U.S. Pat. No. 5,310,667 by substituting an alanine residue for a glycine residue at between positions 80 and 120. Double mutants are also described at U.S. Pat. Nos. 6,225,114 and 5,866,775 in which, in addition to the above mutation, a second mutation (a threonine residue for an alanine residue between positions 170 and 210) is introduced into a wild-type EPSPS gene.
Other work resulted in the production of a double mutant EPSPS maize transformation event GA21 through the introduction of a modified maize EPSPS gene bearing mutations at residue 102 (changing threonine to isoleucine) and at residue 106 (changing proline to serine) of the amino acid sequence encoded by GenBank Accession No. X63374 and shown in U.S. Pat. No. 6,566,587 (see sequence identifier number 3 in the '587 patent) and U.S. Pat. No. 6,040,497. In FIG. 1 is shown Genbank accession number X63374 nucleotide sequence, which is the corn EPSPS nucleotide sequence (SEQ ID NO: 1). The amino acid sequence encoded is set forth beneath the nucleotide sequence (SEQ ID NO: 2). Note that an implied ATG start codon is not included at the beginning of X63374 nucleotide sequence. The double mutant sequence is that in which residue 102 of SEQ ID NO: 2 is changed to isoleucine and residue 106 is changed to serine, and the resulting double mutant protein is SEQ ID NO: 3.
In U.S. Pat. No. 7,045,684, a genomic EPSPS fragment was isolated from maize and subsequently two mutations were introduced into the corn EPSPS gene which resulted in the same mutated EPSPS protein as above, in event GA21. Using the corn EPSPS gene, Genbank accession number X63374, as a probe, a 6.0 kb genomic fragment was isolated, that fragment shown here in FIGS. 2A and 2B and is SEQ ID NO: 4. Two mutations were introduced into this nucleotide sequence; the first a cytosine to thymine substitution at nucleotide 2886, and the second a cytosine to thymine substitution at nucleotide 2897 (the positions are in bold and underlined in FIGS. 2A and 2B and the mutated nucleotide sequence is SEQ ID NO: 5). This resulted in an encoded mutant amino acid which is shown in FIGS. 3A and 3B and is SEQ ID NO: 6 with the residue at position 164 (position 102 of the amino acid of X63374/SEQ ID NO: 3) changed from threonine to isoleucine (Thr to Ile) and at position 168 (position 106 of the amino acid of X63374/SEQ ID NO: 3) changed from proline to serine (Pro to Ser). The resulting mutated amino acid sequence was glyphosate resistant.
The mutated nucleotide sequence of SEQ ID NO: 5 includes the native corn EPSPS promoter, coding region (containing the two mutations), introns and 3′ terminator region. The GA21 event, supra, on the other hand, used a rice actin promoter (McElroy et al. (1990) Plant Cell 2:163-171) and nos terminator (Depicker et al., (1982) Mol. and Appl. Genet. 1:561-573). However, both coding sequences essentially produce the same mutated protein having the change of threonine to isoleucine at position 102 of the protein and proline to serine at position 106 of the protein.
There is a need to identify antibodies that are immunoreactive with the double mutant EPSPS proteins described above so that plants containing such mutated EPSPS proteins can be readily identified. Especially useful would be an antibody that immunoreacts with the double mutant EPSPS protein containing the mutations at residue 102 (Thr to Ile) and at position 106 (Pro to Ser) and is not reactive with the CP4 enzyme, a version used in various commercial glyphosate resistant products, nor with the wild-type EPSPS protein. A method that would avoid time-consuming lab steps would reduce costs, allowing for quick identification of the transgenic plants containing the mutant protein, aiding in breeding and selection. Furthermore, antibodies that are immunoreactive with such proteins could be useful in isolating and purifying the proteins.